Muscarinic receptor regulation of Ca2+ mobilization in a human salivary cell line (original) (raw)
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Ca2+-dependent potentiation of muscarinic receptor-mediated Ca2+ elevation
Cell Calcium, 2004
Muscarinic receptor-mediated increases in Ca 2+ in SH-SY5Y neuroblastoma cells consist of an initial fast and transient phase followed by a sustained phase. Activation of voltage-gated Ca 2+ channels prior to muscarinic stimulation resulted in a several-fold potentiation of the fast phase. Unlike the muscarinic response under control conditions, this potentiated elevation of intracellular Ca 2+ was to a large extent dependent on extracellular Ca 2+ . In potentiated cells, muscarinic stimulation also activated a rapid Mn 2+ entry. By using known organic and inorganic blockers of cation channels, this influx pathway was easily separated from the known Ca 2+ influx pathways, the store-operated pathway and the voltage-gated Ca 2+ channels. In addition to the Ca 2+ influx, both IP 3 production and Ca 2+ release were also enhanced during the potentiated response. The results suggest that a small increase in intracellular Ca 2+ amplifies the muscarinic Ca 2+ response at several stages, most notably by unravelling an apparently novel receptor-activated influx pathway.
Naunyn-Schmiedeberg's Archives of Pharmacology, 1995
We have compared muscarinic acetylcholine receptor (mAChR) coupling to phospholipase C (PLC) and increases in cytoplasmic Ca 2+ concentration [Ca2+]i in human embryonic kidney (HEK) cells, stably expressing either the human m3 or m2 receptor subtype. In m3 mAChR-expressing cells, carbachol stimulated inositol phosphate (InsP) formation and increased [Ca 2 +]i with ECso values of about 2 ~tM and 30 nM, respectively. Maximal inositol 1,4,5-trisphosphate (InsP3) production (about fourfold) was rapid (15 s) and stable for 2rain. Maximal increases in [Ca2+]i were 300.350nM and mainly, almost 90%, due to influx of extracellular Ca 2+. The efficacy of pilocarpine for stimulating InsP and Ca 2 + responses was not significantly different from that of carbachol. All m3 mAChR-mediated responses were pertussis toxin (PTX)-insensitive. In m2 mAChR-expressing cells, carbachol stimulated InsP formation and increased [Ca2+]i with ECso values of about 20 gM and 7 gM, respectively. Maximal InsP formation was only 10-15% of that observed in m3 mAChR-expressing cells, whereas maximal elevations of [Ca 2 +]i were sinailar in both cell types. Formation of InsP3 was rapid (15 s to 2 min) and about twofold above basal. In contrast to m3 mAChR activation, [-Ca2+li increases induced by m2 mAChR activation were exclusively due to Ca 2+ mobilization from intracellular stores. The efficacy of pilocarpine for stimulating InsP and Ca 2 + responses was 50% and 20% of the efficacy of carbachol, respectively. PTX treatment did not affect m2 mAChR-induced PLC stimulation, but reduced the m2 mAChR-mediated increases in [Ca 2 +]i to 50%. In con
Activation of muscarinic receptors in PC12 cells. Stimulation of Ca2+ influx and redistribution
The Biochemical journal, 1986
Ca2+ homoeostasis was investigated in pheochromocytoma neurosecretory (PC12) cells both before and after treatment with nerve growth factor, which induces a neuronal-like differentiation accompanied by a large increase in the number of muscarinic receptors. The resting concentration of free cytosolic Ca2+, [Ca2+]i, measured by the quin2 technique, was found to be higher and more variable in differentiated cells. Moreover, the [Ca2+]i rises induced by the Ca2+ ionophore ionomycin and by depolarizing concentrations of KC1 were greater and more transient. Exposure to carbachol induced modest, but long-lasting, [Ca2+]i rises, which were faster and greater in differentiated than in non-differentiated cells. These effects were due to the activation of the muscarinic receptor, because they were unaffected by nicotinic blockers (hexamethonium and D-tubocurarine) and completely eliminated by low concentrations of the muscarinic antagonists atropine and pirenzepine [IC50 (concn. causing 50% i...
FEBS Letters, 1996
nic m3 receptor plays a dominant role in the regulation of Abstract The m2 and m3 muscarinic acetylcholine receptors Ca 2+ influx in CHO cells. were expressed in CHO cells and were shown to couple to the release of calcium from intracellular stores. The m3 receptor, but not the m2 receptor, also coupled to calcium influx. Chimeric m21 2. Materials and methods m3 receptors were used to determine the structural domain of the m3 receptor linked to the regulation of calcium influx. It was 2.1. Chimeric eDNA construct preparation and cell culture found that the third intracellular loop of m3 receptor plays a
Proceedings of the National Academy of Sciences, 1992
Receptor-mediated changes in cytoplasmic calcium concentrations occur either through release from intracellular calcium stores or by the opening of channels in the plasma membrane, allowing influx of calcium from the extracellular fluid. Carbachol, a muscarinic receptor agonist, stimulated both calcium influx and inositol 1,4,5-trisphosphate (InsP3)-mediated intracellular calcium release in A9 fibroblast cells expressing a m3 muscarinic receptor done. The calcium influx persisted even after pretreatment of cells with phorbol 12-myristate 13-acetate, which completely prevented the rise in inositol phosphates and intracellular calcium levels. The calcium influx was blocked by divalent cations but was not
European Journal of Pharmacology: Molecular Pharmacology, 1991
Muscarinic receptor-linked Ca 2+ mobilization and changes in cyclic AMP were studied in SH-SY5Y and IMR 32 human neuroblastoma celt lines. Muscarinic agonists acetylcholine, carbachol, methacholine and muscarine induced an increase in cytosolic free Ca -,+ in a pertussis toxin (100 ng/ml)-inscnsitive manner in both cell lines. The EDso values in IMR 32 cells (8-98 ,aM) were one order of magnitude higher than in SH-SY5Y cells (0.3-1.6 gM). Oxotremorine and pilocarpine failed to mobilize Ca -'+ in IMR 32 cells. Pirenzepine antagonized carbachol-induced Ca 2+ mobilization in SH-SY5Y cells with a K i value in the range of 150-189 nM whereas the corresponding values in IMR 32 cells were 24-28 riM. Atropine inhibited a carbachol-stimulated increase in cytosolic Ca -,+ with an equal potency in both cell lines (K~ 2-3 nM). Carbachol stimulated cyclic AMP (cAMP) accumulation in SH-SY5Y cells in a pertussis toxin-insensitive manner. In IMR 32 cells carbachol inhibited prostaglandin El-stimulated cAMP accumulation. Treatment of IMR. 32 cells with pertussis toxin abolished the inhibition of stimulated cAMP accumulation. These results suggest that in SH-SY5Y cells the M 3 muscarinic receptor couples to both Ca 2+ mobilization and stimulation of cAMP accumulation. In IMR 32 ceils the M~ receptor seems to couple to Ca 2+ mol~i!ization whereas the inhibition of stimulated cAMP accumulation is coupled to a non-M~ subtype by an inhibitory G-protein.
The EMBO Journal, 1998
Several types of transmembrane receptors regulate cellular responses through the activation of phospholipase C-mediated Ca 2ϩ release from intracellular stores. In non-excitable cells, the initial Ca 2ϩ release is typically followed by a prolonged Ca 2ϩ influx phase that is important for the regulation of several Ca 2ϩsensitive responses. Here we describe an agonist concentration-dependent mechanism by which m3 muscarinic acetylcholine receptors (mAChRs) differentially regulate the magnitude of the release and influx components of a Ca 2ϩ response. In transfected Chinese hamster ovary cells expressing m3 mAChRs, doses of the muscarinic agonist carbachol ranging from 100 nM to 1 mM evoked Ca 2ϩ release responses of increasing magnitude; maximal Ca 2ϩ release was elicited by the highest carbachol concentration. In contrast, Ca 2ϩ influx was maximal when m3 mAChRs were activated by moderate doses (1-10 μM) of carbachol, but substantially reduced at higher agonist concentrations. Manipulation of the membrane potential revealed that the carbachol-induced Ca 2ϩ influx phase was diminished at depolarized potentials. Importantly, carbachol doses above 10 μM were found to couple m3 mAChRs to the activation of an inward, monovalent cation current resulting in depolarization of the cell membrane and a selective decrease in the influx, but not release, component of the Ca 2ϩ response. These studies demonstrate, in one experimental system, a mechanism by which a single subtype of G-protein-coupled receptor can utilize the information encoded in the concentration of an agonist to generate distinct intracellular Ca 2ϩ signals.
BMC Research Notes
Objectives: We examined whether two G protein-coupled receptors (GPCRs), muscarinic M 1 receptors (M 1 Rs) and dopaminergic D 2 receptors (D 2 Rs), utilize endogenously released fatty acid to inhibit L-type Ca 2+ channels, Ca V 1.3. HEK-293 cells, stably transfected with M 1 Rs, were used to transiently transfect D 2 Rs and Ca V 1.3b with different Ca V β-subunits, allowing for whole-cell current measurement from a pure channel population. Results: M 1 R activation with Oxotremorine-M inhibited currents from Ca V 1.3b coexpressed with α 2 δ-1 and a β 1b , β 2a , β 3 , or β 4-subunit. Surprisingly, the magnitude of inhibition was less with β 2a than with other Ca V β-subunits. Normalizing currents revealed kinetic changes after modulation with β 1b , β 3 , or β 4 , but not β 2a-containing channels. We then examined if D 2 Rs modulate Ca V 1.3b when expressed with different Ca V β-subunits. Stimulation with quinpirole produced little inhibition or kinetic changes for Ca V 1.3b coexpressed with β 2a or β 3. However, quinpirole inhibited N-type Ca 2+ currents in a concentration-dependent manner, indicating functional expression of D 2 Rs. N-current inhibition by quinpirole was voltage-dependent and independent of phospholipase A 2 (PLA 2), whereas a PLA 2 antagonist abolished M 1 R-mediated N-current inhibition. These findings highlight the specific regulation of Ca 2+ channels by different GPCRs. Moreover, tissue-specific and/or cellular localization of Ca V 1.3b with different Ca V β-subunits could fine tune the response of Ca 2+ influx following GPCR activation.
Calcium involvement in the muscarinic response of the gastric parietal cell
Cellular Signalling, 1990
The influence of extracellular Ca 2+ on the mediation of carbachol stimulation in isolated rabbit gastric parietal cells was studied. Removing Ca :+ from extracellular medium caused a 42% decrease of the aminopyrine accumulation due to carbachol with the same ECs0 value (~ 5/aM). A short time depletion in extracellular calcium suppressed the carbachol-dependent Ca 2+ influx without affecting Ca :+ release from internal stores (fura-2 measurements). Similarly, the production of inositol phosphates under cholinergic stimulation was reduced by 29%. A rapid increase in Ins(1,4,5)P3 was obtained 5 s after carbachol stimulation, and this increase was not changed in Ca:+-depleted medium. In contrast, a 20 min incubation with carbachol caused a 50% reduction in both basal and carbachol-stimulated inositol phosphate accumulations. In conclusion, phospholipase C activation, intracellular Ca 2+ release and aminopyrine accumulation were sequentially observed following carbachol stimulation of the isolated gastric parietal cell and extraceUular calcium contributed to sustain this acid secretory response.
Calcium and the muscarinic receptor
General Pharmacology: The Vascular System, 1976
Pharmacological receptors may be viewed as composed of two linked functions, a recognition site through which the specificity and selectivity of ligand action is expressed and an amplification or catalytic site which translates the ligand-recognition site interaction into response. The successful elucidation of receptor mechanisms requires analysis of both sites. The muscarinic cholinergic receptors of guinea-pig ileal longitudinal smooth muscle will be discussed from this two function stand point and the source and utilization of Ca 2÷ in excitation-contraction coupling discussed.